Star-PAP controls oncogene expression through primary miRNA 3'-end formation to regulate cellular proliferation and tumour formation.

Star-PAP is a non-canonical poly(A) polymerase that is down regulated in breast cancer. While Star-PAP down regulation impairs target mRNA polyadenylation, paradoxically, we see up regulation of a large number of oncogenes on Star-PAP knockdown. Using two breast cancer cells (MCF7 with high Star-PAP, and MDA-MB-231 with negligible Star-PAP level), we discover that Star-PAP negatively regulates oncogene expression and subsequently cellular proliferation. This regulation is compromised with Star-PAP mutant of 3'-end processing function (serine 6 to alanine, S6A phospho-mutation). Concomitantly, xenograft mice model using MDA-MB-231 cells reveals a reduction in the tumour formation on ectopic Star-PAP expression that is ameliorated by S6A mutation. We find that Star-PAP control of target oncogene expression is independent of Star-PAP-mediated alternative polyadenylation or target mRNA 3'-end formation. We demonstrate that Star-PAP regulates target oncogenes through cellular miRNAs (miR-421, miR-335, miR-424, miR-543, miR-205, miR-34a, and miR-26a) that are down regulated in breast cancer. Analysis of various steps in miRNA biogenesis pathway reveals that Star-PAP regulates 3'-end formation and synthesis of primary miRNA (host) transcripts that is dependent on S6 phosphorylation thus controlling mature miRNA generation. Using mimics and inhibitors of two target miRNAs (miR-421 and miR-424) after Star-PAP depletion in MCF7 or ectopic expression in MDA-MB-231 cells, we demonstrate that Star-PAP controls oncogene expression and cellular proliferation through targeting miRNAs that regulates tumour formation. Our study establishes a novel mechanism of oncogene expression independent of alternative polyadenylation through Star-PAP-mediated miRNA host transcript polyadenylation that regulates breast cancer progression.

Proto-oncogene cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program in the heart and controls cardiac hypertrophy.

AIMS: RBM10 is a well-known RNA binding protein that regulates alternative splicing in various disease states. We have shown a splicing-independent function of RBM10 that regulates heart failure. This study aims to unravel a new biological function of RBM10 phosphorylation by proto-oncogene cSrc that enables anti-hypertrophy gene program and controls cardiac hypertrophy. MATERIALS AND METHODS: We employ in vitro and in vivo approaches to characterise RBM10 phosphorylation at three-tyrosine residues (Y81, Y500, and Y971) by cSrc and target mRNA regulation. We also use isoproterenol induced rat heart and cellular hypertrophy model to determine role of cSrc-mediated RBM10 phosphorylation. KEY FINDINGS: We show that RBM10 phosphorylation is induced in cellular and animal heart model of cardiac hypertrophy and regulates target mRNA expression and 3'-end formation. Inhibition of cSrc kinase or mutation of the three-tyrosine phosphorylation sites to phenylalanine accentuates myocyte hypertrophy, and results in advancement and an early attainment of hypertrophy in the heart. RBM10 is down regulated in the hypertrophic myocyte and that its re-expression reverses cellular and molecular changes in the myocyte. However, in the absence of phosphorylation (cSrc inhibition or phospho-deficient mutation), restoration of endogenous RBM10 level in the hypertrophic heart or ectopic re-expression in vitro failed to reverse cardiomyocyte hypertrophy. Mechanistically, loss of RBM10 phosphorylation inhibits nuclear localisation and interaction with Star-PAP compromising anti-hypertrophy gene expression. SIGNIFICANCE: Our study establishes that cSrc-mediated RBM10 phosphorylation arbitrates anti-hypertrophy gene program. We also report a new functional regulation of RBM10 by phosphorylation that is poised to control heart failure.

Alternative polyadenylation: An enigma of transcript length variation in health and disease.

Alternative polyadenylation (APA) is a molecular mechanism during a pre-mRNA processing that involves usage of more than one polyadenylation site (PA-site) generating transcripts of varying length from a single gene. The location of a PA-site affects transcript length and coding potential of an mRNA contributing to both mRNA and protein diversification. This variation in the transcript length affects mRNA stability and translation, mRNA subcellular and tissue localization, and protein function. APA is now considered as an important regulatory mechanism in the pathophysiology of human diseases. An important consequence of the changes in the length of 3'-untranslated region (UTR) from disease-induced APA is altered protein expression. Yet, the relationship between 3'-UTR length and protein expression remains a paradox in a majority of diseases. Here, we review occurrence of APA, mechanism of PA-site selection, and consequences of transcript length variation in different diseases. Emerging evidence reveals coordinated involvement of core RNA processing factors including poly(A) polymerases in the PA-site selection in diseases-associated APAs. Targeting such APA regulators will be therapeutically significant in combating drug resistance in cancer and other complex diseases. This article is categorized under: RNA Processing > 3' End Processing RNA in Disease and Development > RNA in Disease Translation > Regulation.

A Splicing-Independent Function of RBM10 Controls Specific 3' UTR Processing to Regulate Cardiac Hypertrophy.

RNA binding motif protein 10 (RBM10) is a regulator of alternative splicing in apoptosis and inflammation. We discovered a splicing-independent function of RBM10 critical for the regulation of heart failure (HF). RBM10 is enriched in the heart and associates with Star-PAP (TUT1) to control the expression and 3' end processing of cardiac mRNAs. The RBM10 RRM2 domain binds the Star-PAP catalytic domain, which directs Star-PAP activity toward polyadenylation. RBM10 binds the pre-mRNA UTR, assembles the Star-PAP complex, and guides this complex specifically to mRNAs encoding anti-hypertrophy regulators. Accordingly, we tested cellular hypertrophy in rat cardiomyoblasts and cardiac hypertrophy (CH) and the subsequent progression to HF in Wistar rat hearts. We demonstrated downregulation of RBM10 during CH and HF. Ectopic re-expression of RBM10 rescued cardiomyocyte hypertrophy. RBM10 depletion evoked a hypertrophic response in H9c2 cells. Our results establish an anti-hypertrophy mechanism mediated by RBM10 in the heart that is directly linked to HF.

Nuclear Phosphatidylinositol-Phosphate Type I Kinase α-Coupled Star-PAP Polyadenylation Regulates Cell Invasion.

Star-PAP, a nuclear phosphatidylinositol (PI) signal-regulated poly(A) polymerase (PAP), couples with type I PI phosphate kinase α (PIPKIα) and controls gene expression. We show that Star-PAP and PIPKIα together regulate 3'-end processing and expression of pre-mRNAs encoding key anti-invasive factors (KISS1R, CDH1, NME1, CDH13, FEZ1, and WIF1) in breast cancer. Consistently, the endogenous Star-PAP level is negatively correlated with the cellular invasiveness of breast cancer cells. While silencing Star-PAP or PIPKIα increases cellular invasiveness in low-invasiveness MCF7 cells, Star-PAP overexpression decreases invasiveness in highly invasive MDA-MB-231 cells in a cellular Star-PAP level-dependent manner. However, expression of the PIPKIα-noninteracting Star-PAP mutant or the phosphodeficient Star-PAP (S6A mutant) has no effect on cellular invasiveness. These results strongly indicate that PIPKIα interaction and Star-PAP S6 phosphorylation are required for Star-PAP-mediated regulation of cancer cell invasion and give specificity to target anti-invasive gene expression. Our study establishes Star-PAP-PIPKIα-mediated 3'-end processing as a key anti-invasive mechanism in breast cancer.

Distinct regulation of alternative polyadenylation and gene expression by nuclear poly(A) polymerases.

Polyadenylation of nascent RNA by poly(A) polymerase (PAP) is important for 3' end maturation of almost all eukaryotic mRNAs. Most mammalian genes harbor multiple polyadenylation sites (PASs), leading to expression of alternative polyadenylation (APA) isoforms with distinct functions. How poly(A) polymerases may regulate PAS usage and hence gene expression is poorly understood. Here, we show that the nuclear canonical (PAPα and PAPγ) and non-canonical (Star-PAP) PAPs play diverse roles in PAS selection and gene expression. Deficiencies in the PAPs resulted in perturbations of gene expression, with Star-PAP impacting lowly expressed mRNAs and long-noncoding RNAs to the greatest extent. Importantly, different PASs of a gene are distinctly regulated by different PAPs, leading to widespread relative expression changes of APA isoforms. The location and surrounding sequence motifs of a PAS appear to differentiate its regulation by the PAPs. We show Star-PAP-specific PAS usage regulates the expression of the eukaryotic translation initiation factor EIF4A1, the tumor suppressor gene PTEN and the long non-coding RNA NEAT1. The Star-PAP-mediated APA of PTEN is essential for DNA damage-induced increase of PTEN protein levels. Together, our results reveal a PAS-guided and PAP-mediated paradigm for gene expression in response to cellular signaling cues.